Scleroderma (Systemic Sclerosis, SSc) is an idiopathic autoimmune disease characterized by cutaneous and visceral fibrosis in which many patients are affected by interstitial lung disease (SSc-ILD) which lacks specific, highly efficacious therapy. The response of SSc-ILD to immunomodulatory agents and in some cases bone marrow transplantation suggests involvement of leukocytes in disease pathogenesis. Detailed examination of lung tissue obtained from patients with SSc-ILD frequently reveals inflammatory cells juxtaposed with normal and fibrotic extracellular matrix (ECM). It is therefore notable that fibrocytes, a population of leukocytes displaying mesenchymal characteristics, have in several studies demonstrated enhanced accumulation in the SSc-ILD blood and/or lung. The significance of these cells and the factors regulating their appearance in this clinical context remains unknown. Published and preliminary work from our laboratory indicates the laminin-like neuroimmune molecule Netrin-1 (NTN-1) is important in this setting. NTN-1 stimulates cellular attraction via binding to its attractive receptor, Deleted in Colorectal Cancer-1 (DCC-1) while cellular repulsion and invasion is driven by interactions with its repulsive receptor, Uncoordinated-5a (UNC5a). We explored the relevance of these pathways to SSc-ILD using a novel translational platform based on decellularized human lungs to demonstrate that the accumulation of collagen-producing leukocytes is regulated by biochemical and mechanical aspects of the human lung ECM, and that SSc-ILD PBMCs produce more fibrocytes when exposed to the digestible components of the fibrotic Scleroderma lung. NTN-1 expression is increased on CD14lo monocytes in SSc-ILD where it regulates leukocyte-matrix interactions and TGF-?1 secretion by PBMCs. Last, studies performed in the bleomycin model of pulmonary fibrosis demonstrate that partial deficiency of NTN-1 ameliorates histologic and biochemical readouts of fibrosis, TGF-?1 production, and the accumulation of CD45+ColI?1+ cells, indicating that NTN-1 neutralization might be a therapeutic target in this context. Exploration of these areas has the potential to advance the understanding of lung involvement in SSc. Therefore, in Aim 1, we will use standard immunolocalization techniques and multiparametric mass cytometry to define the expression of NTN-1, DCC-1, and UNC5a on innate and adaptive immune cells in the lungs and blood of two well characterized longitudinal cohort of subjects with SSc-ILD. In Aim 2, we use neutralizing antibodies and virally mediated overexpression strategies combined with mechanically tunable polymer networks rendered bioactive through conjugation with normal and SSc-ILD lung matrix to determine whether Netrin-1's modulation of fibrocyte accumulation results from receptor mediated interactions with ECM components or from the combinatorial influence of the protein composition and stiffness of the underlying substrate. In Aim 3, we use mice with null mutations of UNC5a and DCC-1, as well as mice with cell specific deletion of NTN-1 and TGF-?1 to determine their contribution to fibrosis and intrapulmonary fibrocyte accumulation seen in two separate models of experimentally-induced lung fibrosis.